The main objective of this research remains to investigate the biochemical properties and hormonal regulatory mechanisms of cyclic nucleotide phosphodiesterases. The direction of these studies is to provide an integrated, systematic, and detailed analysis of the function of cyclic nucleotide phosphodiesterases in the processes of growth, differentiation, and hormone action. Linear sucrose gradients, ion-exchange chromatography, isoelectric focusing, and gel filtration are the primary fractionation techniques to be used for enzyme separation, identification, and comparison. Cultures of baby hamster kidney fibroblasts (BHK 21 c/13) and purified human peripheral blood lymphocytes will be used to test the hypothesis that activation of specific forms of phosphodiesterase activity involves the mobilization and/or synthesis of factors as a consequence, of interactions of hormones and their cellular receptors. Analytical and preparative techniques will be used to establish the existence of factors produced by hormonal stimulation. We plan continued analyses of protein synthetic dependent and independent mechanisms of activation of high affinity cyclic nucleotide phosphodiesterases by serum, insulin, methylxanthines, and plant lectins such as concanavalin A and phytohemagglutinin will be studied in these cells. Knowledge gained from these investigations will be applied towards studies of hormone modulation of cyclic nucleotide phosphodiesterase activity in adipose and hepatic tissues. We plan a major emphasis during the next year to purify dog kidney cyclic nucleotide phosphodiesterase to homogeneity on a preparative scale and to continue biochemical and immunological characterization of these enzyme forms.